T H E AMKHICAN JOURNAL OK CLINICAL PATHOLOGY
VIII. II. N». 1, pp. 22-'.'(!
.lnmiurv, 1964
Cuiiyrmlii i- 1%I by Tlic Williams & Wilkins Co.
J'rintcd in tr'.S.A.
DETERMINATION OF URINARY COPPER BY MEANS OF DIRECT
EXTRACTION WITH ZINC DIBEXZYL DITHIOCARBAMATE
ANTHONY J. GIORGIO, M.D., GEORGE E. CARTWRIGHT, M.D., AND
MAXWELL M. W1XTROJ3E, M.D., P H . D .
Department of Medicine, University of Utah, College of Medicine, Salt Lake City, Utah
The analysis of copper in urine by means
of the methods currently in use 2 , 5 requires
concentration of the urine and wet ashing
of the concentrate with nitric, sulfuric, and
perchloric acids. The disadvantages of
ashing methods are that time and skill are
required of the analyst, the acids may
introduce significant amounts of copper, and
loss of copper owing to volitalization and
frothing may occur. Unless extreme precautions are taken, the danger of an
explosion is always present when perchloric
acid is used.
In the method described in this paper,
concentration of the urine and wet ashing
are avoided by means of extracting the
copper directly from acidified urine into a
solution of zinc dibenzyl dithiocarbamate
(DBDC) in carbon tetrachloride. The
stable colored complex of copper DBDC
which is formed is soluble in carbon tetrachloride.
The use of DBDC as a copper colorimetric
reagent was first reported by Martens and
Githens in 1952.6 These workers used the
reagent to analyze for copper in certain
rubber and dye chemicals. They reported
that the reagent was both stable and
specific for copper. Recovery of 5 ng. of
copper in the presence of 0.5 Gra. of Fe 11 ,
Fe 1 ", Ca, As 1 ", As v , Zn, Cr, Cd, Al, or Mn,
ranged from 4.6 to 5.5 ^g- The presence of
Ag, Hg. Xi, and Co in amounts of 0.1 to
5 mg. also did not interfere with the
determination of 5 /ig. amounts of Cu.
These authors reported some interference
from Bi, but inasmuch as this metal is
seldom found in urine in more than trace
quantities, 8 it is of no concern regarding
the present application.
Since the original report of Martens and
Githens, the DBDC reagent has been used
to measure the amount of copper in acid
digests of enzyme homogenates13 and urine.7
The method has also been used for the direct
extraction of copper from beer.11
METHOD
Preparation of Glassware
All glassware was first immersed in
dichromate-sulfuric acid solution for 45
min., rinsed, and then immersed in concentrated hydrochloric acid for 30 min.
After 3 rinses with demineralized water it
was rinsed in 1 per cent disodium ethylenediaminetetraacetate. The vessels were finally
rinsed 12 times in demineralized water and
oven-dried.
Reagerits
DBDC. This reagent was obtained from
the Naugatuck Chemical Co. of Naugatuck,
Connecticut, under the trade name of
Arazate. A 0.015 per cent solution in carbon
tetrachloride was prepared and stored in
clear glass bottles.
Demineralized watei- (pH 5.4)- Distilled
water was passed through a Deeminizer
(Crystal Research Laboratories, Hartford,
Connecticut).
Copper-free water (pH 6.8). Deeminized
water was passed through a 40- by 2-cm.
column of Dowex A-l, 50-100 mesh, cation
exchange resin in the ammonium form
(Dow Chemical Co., Midland, Michigan) at
a flow rate of 2 ml. per sq. cm. per min.
Carbon tetrachloride. Carbon tetrachloride
(low sulfur, A.R. Grade) was obtained from
Received, June 10, 19G3; accepted for publication October 3.
Dr. Giorgio is Instructor in Medicine; Dr.
Cartwright is Professor of Medicine; and Dr.
Wintrobe is Professor and Head, Department of
Medicine.
This investigation was supported by a research
grant (AM-044S9) and a graduate training grant
(2A-50'JS) from the National Institute of Arthritis
and Metabolic Diseases, Department of Health,
Education, and Welfare, Bethesda, Maryland.
22
Jan.1964
23
U1UXE COITEU
Mallinckrodt Chemical Works, St. Louis,
Missouri.
Copper standard. A stock solution containing 100 mg. of "Spec pure" copper
(Johnson & Matthcy Co., London, England)
in 10 ml. of isothennaHy purified10 5 N IIC1
was prepared. A working standard (pll 3.5)
containing 2 ng. of copper per ml. was
prepared by means of dilution of the stock
solution with dcmineralized water. Copperfree water was not used because the higher
pll might have reduced the stability of the
dilute standard. 10
Procedure
Collection of wt'ne. Urine was collected in
soft glass 2\ 2-liter bottles. Males urinated
directly into the bottles. Females urinated
into 600- to 800-ml. beakers, then transferred the specimen to the bottle.
Preservatives were not used except in the
Wilson's disease collections, to which 5 ml.
of toluene was added.
Extraction. Two 10-ml. aliquots of urine
were pipeted into separate 125-ml. pearshaped separately funnels. One milliliter
of concentrated hydrochloric acid was
added to each, followed by brief swirling.
After standing for 15 min., 10 ml. of the
DBDC reagent was added to one; and to
the other, referred to as the extraction
blank, 10 ml. of carbon tetrachloride was
added.
A copper reference standard funnel was
prepared simultaneously with each set of
urine determinations. One milliliter of the
2 fig. per ml. copper standard was added to
10 ml. of copper-free water. One milliliter
of hydrochloric acid and 10 ml. of the
DBDC reagent were added, and the funnel
was allowed to stand for 15 min.
The funnels were then briskly shaken by
hand for 1 min. After separation, the lower
carbon tetrachloride layer was drawn off
and centrifuged at 2000 g for 10 min. in
order to achieve maximal clarity prior to
spectrophotometry.
Acidification and heating prior to extraction were found to enhance the recovery of
both native and added copper from urine
specimens containing bilirubin. Two 10-ml.
urine aliquots were placed in 15-ml. centri-
fuge tubes.and acidified with 1 ml. of hydrochloric acid. They were then heated at !)-l C.
in a water bath for 15 min. After cooling,
the urines were placed in soparatory funnels
and again acidified with 1 ml. of hydrochloric acid. Extraction was performed as
before.
Spectrophotometry and calculation. Optical
densities (O.D.) were measured at wave
length 435 lru* in a Beckman D.U. spectrophotometer with 2.5 ml. cuvets with a light
path of 50 mm. The unknown solution and
the standard solution were read against the
DBDC reagent which was set at zero O.D.
The extraction blank was read against
carbon tetrachloride which was set at
zero O.D.
Calculation of copper concentration was as
follows:
Concentration of copper standard (^g./ml.)
y O.D. unknown — O.D. extraction blank
O.D. of Cu standard
= /ig. copper in 10 nil. of urine
Wet ashing. Ten milliliters of urine was
pipeted into a 100-ml. Kjeldahl flask. Five
milliliters of concentrated nitric and 1 ml. of
concentrated sulfuric acid were added, along
with 5 glass beads. The flasks were capped
and allowed to stand overnight in order to
minimize frothing upon heating.
The contents were heated until charring
occurred. When cooled, 5 ml. of concentrated
nitric acid and 1 ml. of concentrated perchloric acid were added, and the digestion
resumed for 20 min. Twenty milliliters of
copper-free water and 2 ml. of ammonium
hydroxide were added to the clear digests
after they had cooled to room temperature.
The diluted digests were then extracted with
10 ml. of the DBDC reagent. A reagent
blank and a standard solution were run
along with the urine sample. The concentration of copper was calculated by reading the
O.D. of the standard solution and the
unknown solution against the reagent blank.
EXPERIMENTAL STUDIES
Completeness of extraction. In order to
study the completeness of copper extraction,
10-ml. solutions of copper ranging in
24
GIORGIO ET AL,
concentration from 0.1 Mg- to 20 Mg- per
ml. were extracted with 10 ml. of the
DBDC reagent. Extraction was performed
as described for the urine determinations.
All of the copper was extracted by the
DBDC carbon tetrachloride solution from
the solutions containing 10 Mg- of copper per
ml. or less. A second extraction with the
solution was required to remove all of ,the
copper when the concentration was greater
than 10 Mg- of copper per ml.
Rapidity of color development and stability
of the copper complex. Ten milliliters of an
0.2 Mg- Cu per ml. solution was extracted
with the DBDC reagent and the O.D. of the
colored complex was determined immediately after centrif ligation and at
various intervals up to 24 hr. Initial and
24-hr. readings were 0.277 and 0.276,
respectively. The O.D. measured at ]/i,
]/2, 1,2, and 4 hr. varied less than 1 per cent
from these figures.
A solution containing 0.5 Mg- of copper per
ml. was extracted and the initial O.D. of the
complex was 0.635. The complex was then
stored in a stoppered, clear glass vessel and
exposed to room light for 1 month, after
which time the O.D. reading was 0.641.
Stability of reagent. Ten milliliters of a
solution containing 0.2 Mg. of Cu per ml. was
extracted with freshly prepared DBDC
carbon tetrachloride solution. The reagent
was then left standing in a clear bottle for
1 month and another aliquot of the above
copper solution was extracted. The O.D.
readings were 0.269 and 0.266, respectively.
Recovery of copper added to urine.
One-, 2 - , and 5-Mg- amounts of copper
were added to 10-ml. aliquots of urine
obtained from normal persons and from
patients with Wilson's disease, obstructive
jaundice, and nephrosis. The patients with
Wilson's disease were receiving o-penicil-.
lamine therapy at the time of the collections.
All samples were analyzed by means of
the direct-extraction method. The results
are summarized in Table 1.
The recovery of the copper added to
normal urine and to the urine from patients
with Wilson's disease ranged from 91 to
117 per cent. The recovery of the copper
added to the urine specimens high in
Vol. 1)1
TABLE 1
RECOVERY OF ADDED COPPER FROM SPECIMENS OF
NORMAL, AND ABNORMAL URINE
Specimen
Urine
Copper
Copper Added
Copper
Recovered
per cent
HI J10 ml.
ui-
Normal
0.09
Normal
0.24
1
2
5
2Mg2 Mg- + 1
mg. Fe
2 Mg. + 5
mg. Fe
2 jug. + 10
mg. Fe
1
2
5
1
2
5
1
2
5
1
2
5
1
2
5
1
2
5
Wilson's disease,*
Case A
12.5
Wilson's disease,*
Case B
9.4
Obstructive jaundice, Case A
2.4
Obstructive jaundice, Case B
1.3
Nephrosis, Case A
1.3
Nephrosis, Case B
1.1
91
117
103
97
97
97
97
103
99
98
102
1C3
101
96
92
83
75
89
88
95
93
93
85
80
75
* Five-milliliter specimen analyzed.
bilirubin or protein ranged from 75 to 96
per cent.
One-, 5 - , and 10-mg. amounts of iron,
prepurified by means of extraction with
DBDC, were added to 10-ml. aliquots of
normal urine containing 2 Mg- of added
copper in order to determine if iron interferes
with. Ui& extraction and measurement of
copper.
No significant interference or color
enhancement was noted. Ninety-seven per
cent of the added copper was recovered
(Table 1).
Comparison of direct extraction method with
the wet ashing method. Duplicate 10-ml.
aliquots of urine were prepared. One aliquot
was analyzed by means of the direct extrac-
Jan. 1964-
25
URINE COITKIl '
tion method. The other aliquot was wet
ashed by means of the method described
and the copper was then extracted with
the DBDC rcagetit. The results obtained
with the two methods are listed in Table 2.
No consistent or significant difference was
noted between the two methods.
Reproducibility of the method. The results
of replicate determinations performed at
intervals of 1 to 10 days on a standard
solution of copper, a normal specimen of
urine, and a specimen of urine from a
patient with Wilson's disease are tabulated
in Table 3.
At a concentration of 0.1 tig. of Cu per
10 ml. the method was reproducible within
± 9 0 per cent; at a concentration of 2 ng. of
Cu per 10 ml. the method was reproducible
within ±G per cent; and at a concentration
of 5 /ig. of Cu per 10 ml. the method was
reproducible within ± 1 2 per cent.
TABLE 2
COMPARISON OF T H E D I R E C T EXTRACTION M E T H O D
WITH THK W E T A S H M E T H O D
Specimen
Person
Tested
Direct
Extraction
A
B
C
D
E
F
G
H
I
J
0.01
0.10
0.17
7.0
5.8
2.4
1.2
1.3
1.1
0.66
Wet Ash
ft- copp tr/10 ml.
Normal
Normal
Normal
Wilson's disease
Wilson's disease
Obstructive jaundice
Obstructive jaundice
Nephrosis
Nephrosis
Nephrosis
0.02
0.15
0.19
7.0
5.5
1.7
0.3
1.7
1.8
0.64
TABLE 3
R E P R O D U C I B I L I T Y OF THE M E T H O D
Specimen
Number
ot Determinations
Mean ± 1 S.D.
(ig. copper/10 ml.
Copper standard (2
10
2.00 ± 0.002
10
10
0.11 ± 0.049
5.07 ± 0.310
fg.)
Normal urine
Urine from patient
with Wilson's disease
Five determinations wore made over the
course of 1!) days on a specimen of normal
urine which was left standing in the collection bottle at 4 C. No significant variation
was noted from the initial value, thus
precluding the bottle as a source of loss of
copper or contamination.
Excretion of urinary copper in normal
adults. A single 24-hr. urine specimen was
collected from each of 10 normal adult
females and 10 normal adult males. The
mean concentration of copper ± 1 standard
deviation (S.D.) for the group was 15 ± 3.7
tig. per liter (7 to 25). Xo difference in
copper excretion was noted between the
sexes. The mean 24-hr. excretion of copper
± 1 S.D. for the group was 21 ± 5.2 pg.
(8 to 57).
DISCUSSION'
The major advantage of this method for
the determination of urine copper is its
simplicity. A number of determinations can
be made in less than 1 hr. and with only
routine laboratory facilities. The development of color with the DBDC reagent is
rapid and the color complex is stable
indefinitely. The only disadvantage of the
method is that precaution is required to
avoid prolonged inhalation of carbon tetrachloride vapors. Other solvents for DBDC
were tried, including methyl-ethyl ketone
and chloroform, but they were found to be
unsuitable because of excessive aqueous
miscibility or extraction of extraneous
colors from urine.
The mean recovery of copper added to
normal urine or to urine obtained from
patients with Wilson's disease was 100 per
cent, with extremes of 91 and 117 per cent.
The mean recovery of copper added to
specimens of urine containing increased
amounts of protein or bilirubin was 87 per
cent, with extremes of 75 to 96 per cent.
Recovery of copper added to normal and to
abnormal specimens of urine has not, to the
authors' knowledge, been reported for the
wet ashing methods. Porter 9 mentions that
1 tig. of copper added to normal urine could
be recovered with a maximal error of 30
per cent. The results with the present
method compare favorably with this value
2G
GIOKGIO ET AL.
and are considerably better than the results
obtained with the semiquantitative, direct
extraction method proposed by Earl. 4
The mean value of 21 y.g. per day for
copper excretion by normal persons obtained
with the present method compares favorably
with the mean values of 9,2 18,3 30,12 48,1 and
5314 Mg- per day reported by others with
wet ashing technics.
SUMMARY
This paper deals with the description and
discussion of a method for determining
copper in urine, by means of directly
extracting the acidified specimen with a
carbon tetrachloride solution of zinc dibenzyl dithiocarbamate (DBDC).
The method is simple to perform and the
DBDC reagent is stable indefinitely.
Development of color occurs rapidly and the
colored copper complex is stable for at
least 1 month. The mean recovery of copper
added to normal urine or to urine obtained
from patients with Wilson's, disease was
100 per cent (91 to 117). The mean recovery
of copper added to specimens of urine
containing increased amounts of protein or
bilirubin was 87 per cent (75 to 96). The
method was reproducible within approximately ± 1 0 per cent, with concentrations
of copper from 2 to 5 y.g. per 10 ml. of urine.
The mean 24-hr. excretion of copper
( ± 1 S.D.) by 20 normal adult persons was
21 ± 5.2 ng.
STJMMARIO IX IXTERLIXGUA
Le presente communication describe e
discute un methodo pro determinar le contento urinari de cupro. Le specimen es
acidificate, e le extraction es effectuate
directemente per medio de un solution, in
tetrachloruro de carbon, de dithiocarbamato
dibenzyljc de zinQ.
Le technica es simple e le reagente es indefinitemente stabile. Le disveloppamento
dc color occurre rapidemente, e le colorate
complexo a cupro es stabile durante al minus
1 mense. Le valor medic del cupro retrovate
post addition, a urina normal o a urina de
patientes con morbo de Wilson esseva 100
pro cento (91 a 117 pro cento). Le valor
medic del cupro retrovate post addition a
Vol. 41
urina continentc augmentate conccntrationes
de protcina o bilirubina esseva 87 pro cento
(75 a 90 pro cento). Le methodo habeva un
reproducibilitatc de intra ± 1 0 pro cento,
con conccntrationes de cupro de 2 a 5 ng. per
10 ml. de urina.
Le valor medie del excretion de cupro in
24 horas (D.S. ± 1 ) per 20 adultos normal
esseva 21 ± 5.2 jug.
Acknowledgment.
We a r e indebted t o M r s .
Margene Yamada for lier patience a n d skill in
preparing the glassware.
REFERENCES
1. B E A R N ,
A.
G.,
AND K U N K E L ,
H.
G.: Ab-
normalities of copper metabolism in Wilson's disease and their relationship to t h e
aminoaciduria. J. Clin. Invest., 3 3 : 400409, 1954.
2. C A R T W R I G H T ,
G.
E.,
GTJBLER,
C.
J . , AND
W I N T R O B E , M . M . : Studies on copper
metabolism. X I . Copper a n d iron metabolism in t h e nephrotic syndrome. J . Clin.
Invest., 33: 0S5-G98, 1954.
3. C H ' E N P E I - E N . : Abnormalities of copper m e -
tabolism in Wilson's disease. Chinese M e d .
J., 75: 917-924, 1957.
4. E A R L , C. J . : Screening t e s t for excessive urinary copper. Lancet, 1: 234-237, 1954.
5. E D E N , A., AND G R E E N , H . H . : M i c r o - d e t e r m i -
nation of copper in biological
Biochem. J . , 34:1202-1208,1940.
material.
0. M A R T E N S , R . I . , AND G I T H E N S , R . E . , S R . :
Small amounts of copper in dves and rubber
chemicals. Anal. Chem., 24: 991-993, 1952.
7. M A Y T U M , W. J . , G O L D S T E I N , N . P . , M C G C C K I N ,
W. F . , AND O W E N , C . A., J R . : Copper m e -
tabolism in Wilson!s disease, Laeunec's
cirrhosis, a n d hemochromatosis: Studies
with radiocopper (Cu 6 4 ). Proc. Staff Meet.
Mayo Clin., 36: 641-060, 1961.
S. M E L T Z E R , L . E . , R U T M A N , J., G E O R G E , P . ,
R U T M A N , R., AND K I T C H E L L , J . R . : T h e
urinary excretion p a t t e r n of trace metals in
diabetes mellitus. A m . J . M e d . S c , 244:
2S2-289, 1902.
9. PORTER, H . : Copper excretion in t h e urine of
normal individuals a n d of patients with
hepatolenticular degeneration
(Wilson's
disease). Arch. Biochem., 3 1 : 202-265, 1951.
10. SANDET.L, E . 13.: Colorimetric Determination
of Traces of Metals, E d . 3 . N e w Y o r k :
Interscience Publishers, 1959, p p . 18-20.
11. S T O N E ,
I.,
ETTINGER,
R.,
AND G A N T Z ,
C:
Nonashing technique for determination of
traces of copper in m a l t beverages. Anal.
Chem., 25: 893-S95, 1953.
12. VAN RAVESTEYN, A. I I . : Metabolism of copper
in m a n . Acta Med. Scand., 118: 103-196,
1944.
13. YONETANI. T . : Studies on cytochrome oxidase. I I I . Improved preparation a n d some
properties. J . Biol. Chem., 236: 1680-1688,
1961.
14. ZIMDAIIL, W. T . , H Y M A N , I . , AND C O O K , E . D . :
Metabolism of copper in hepatolenticular
degeneration. Neurology, 3 : 509-576, 1953.